1 Supplementary Materials 1 2 Supplementary figures 3 4 Supplementary figure 1. Modulation in cytokine production in DSS treated Il33 -/- mice. 5 Quantification of indicated cytokines in the colon explants of WT and Il33 -/- mice at indicated 6 days post DSS administration. Similar data was obtained with the sera (not shown). Data 7 represent two independent experiments and analyzed by Mann-Whitney U test. Error bars 8 represent mean±S.E.M with 10 mice per group per time point. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Day 4 Day 8 0.0 0.5 1.0 1.5 2.0 IP-10 (ng/ml) * NS WT Il33 -/- Day 4 Day 8 0 5 10 15 20 TNF-α (pg/ml) NS NS Day 4 Day 8 0 25 50 75 100 125 IL-17 (pg/ml) NS NS Day 4 Day 8 0 20 40 60 80 CCL5 (pg/ml) NS NS Day 4 Day 8 0.0 0.2 0.4 0.6 0.8 1.0 GM-CSF (ng/ml) NS NS
14
Embed
NS * NS NS IP-10 (ng/ml) WT IL-17 (pg/ml) GM-CSF (ng/ml)10 15 20 25 Il33 Relative expression Water DSS N.D. *** 0 2 4 6 8 10 Il33 Relative expression 0 4 8 Days post DSS *** A B C
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4!Supplementary figure 1. Modulation in cytokine production in DSS treated Il33-/- mice. 5!Quantification of indicated cytokines in the colon explants of WT and Il33-/- mice at indicated 6!days post DSS administration. Similar data was obtained with the sera (not shown). Data 7!represent two independent experiments and analyzed by Mann-Whitney U test. Error bars 8!represent mean±S.E.M with 10 mice per group per time point. 9! 10! 11!
12!
13!
14!
15!
16!
17!
18!
19!
20!
21!
22!
23!
24!
25!
Day 4 Day 80.0
0.5
1.0
1.5
2.0
IP-1
0 (n
g/m
l)
*NSWTIl33-/-
Day 4 Day 80
5
10
15
20
TNF-α
(pg/
ml)
NS NS
Day 4 Day 80
25
50
75
100
125
IL-1
7 (p
g/m
l)
NS NS
Day 4 Day 80
20
40
60
80
CC
L5 (p
g/m
l)
NS NS
Day 4 Day 80.0
0.2
0.4
0.6
0.8
1.0
GM
-CSF
(ng/
ml) NS NS
! 2
26!
Supplementary figure 2. Levels of IL-33 and IL-1α are increased in the colon after DSS 27!administration. WT and Il33-/- mice were treated with DSS in drinking water for 6 days, followed 28!by drinking water for 2 days. (A) Western blot analysis for IL-33 in the colon lysates. (B) qRT-29!PCR for Il33 expression in the colon tissue. (C) qRT-PCR analysis of Il33 and Il1α expression 30!from indicated cell populations from the epithelial (Ep) and colonic lamina propria (cLP) fractions 31!of WT mice at day 8. (D) Western blot analysis for caspase-1 in colon lysates. (E) H&E staining 32!at 40x original magnification of colon sections at day 4 post DSS. Data represent two 33!
Ep Epc
am+
Ep CD45
+
cLP C
D90+
cLP M
HCII+02468
200400
Il1α
R
elat
ive
expr
essi
on *** *** *** ***
Ep Epc
am+
Ep CD45
+
cLP C
D90+
cLP M
HCII+0
5
10
15
20
25
Il33
Rel
ativ
e ex
pres
sion
WaterDSS
N.D.
*** *** ***
0
2
4
6
8
10
Il33
Rel
ativ
e ex
pres
sion
0 4 8Days post DSS
***A B
C
WT Il33-/-
D
E
IL-33 GAPDH
Day 8 Day 0
WT Il33-/- Day 4
caspase-1 p10
caspase-1 p 45
Short Exposure
Long Exposure
Day 8 WT Il33-/-
! 3
independent experiments and analyzed by Mann-Whitney U test. Error bars represent 34!mean±S.E.M with 5 mice per group per time point. 35!
36!
37!
38!
39!
40!
! 4
41!Supplementary figure 3. IL-1α promotes colitis and associated cancer. (A) Survival and (B) 42!disease Activity Index of WT and Il1α-/- mice administered 4% DSS in drinking water. (C) Body 43!weight change of WT and Il1α-/- mice injected with AOM on day 0 and administered 5 rounds of 44!3.5% DSS in drinking water. (D) and (E) Colon histology analysis and (F) proportion of mice with 45!low- or high-grade epithelial dysplasia at day 108 post AOM injection. (G) H&E staining at 10x 46!original magnification of the distal and middle colon sections at day 108 post AOM injection. 47!
Data represent two independent experiments and analyzed by two-way ANOVA followed by 48!Holm-Sidak post test (B and C) or Mann-Whitney U test (D and E). Error bars represent 49!mean±S.E.M. 50!
51!
52!
53!
54!
55!
56!
57!
58!
59!
60!
61!
62!
63!
64!
65!
66!
67!
68!
69!
70!
71!
72!
73!
74!
75!
76!
77!
78!
79!
80!
81!
82!
! 6
83!Supplementary figure 4. Modulation of goblet cells during DSS administration in Il33-/- 84!mice. (A) Quantification of expression of mucins and goblet cell associated genes in the colons 85!of WT and Il33-/- mice at indicated days post DSS administration by qRT-PCR. (B) 86!Quantification of the number of goblet cells per crypt. (C) PAS staining at 10x original 87!magnification of colon sections. Data was analyzed by Kruskal-Wallis test followed by Dunn’s 88!post test. Error bars represent mean±S.E.M. N= 8 each for (A), N=5 each for (B) and (C). Each 89!dot represents an individual crypt. 90!
91!
92!
93!
94!
95!
96!
97!
98!
99!
100!
101!
024
100
200
Muc2
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 40
1
2
3
4Muc1
Rel
ativ
e ex
pres
sion NS NS NS
Day 0 Day 8Day 4
WTIl33-/-
0.0
0.5
1.0
1.5
2.0
Muc3
Rel
ativ
e ex
pres
sion **NS NS
Day 0 Day 8Day 4
012345
Muc4
Rel
ativ
e ex
pres
sion NS NSNS
Day 0 Day 8Day 40
2
4
6
8
Muc5ac
Rel
ativ
e ex
pres
sion ***NS NS
Day 0 Day 8Day 40
2
4
6
Tff3
Rel
ativ
e ex
pres
sion NS NSNS
Day 0 Day 8Day 4
0
10
20
30
Gob
let c
ells
per
cry
pt NS
Day 0 Day 8Day 4
*** ****
A
B
WT
Il33-/-
Day 0 Day 4 Day 8 C
! 7
102!Supplementary figure 5. ILC2 analysis in the colons of WT and Il33-/- mice. (A) 103!Quantification of expression of type2 cytokines in the colons of WT and Il33-/- mice at indicated 104!days post DSS administration by qRT-PCR. (B) Gating strategy for ILC2 cells, where Lin- 105!represents CD3-B220-Ly-6C-Ly-6G-CD11b-Ter119-NKp46-. Proportion and total number of (C) 106!ILC2s and (D) TH2 cells in the lamina propria. Data represent two independent experiments and 107!analyzed by Kruskal-Wallis test followed by Dunn’s post test. Error bars represent mean±S.E.M. 108!with 10 mice per group per time point. 109!
110!
111!
112!
113!
0
5
10
15
Amphiregulin
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 40
1
2
3
4Il5
Rel
ativ
e ex
pres
sion ***NS NS
WTIl33-/-
Day 0 Day 8Day 40
5
10
15
Il13
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 4
0
500
1000
1500
2000
ILC
2 pe
r col
on
Day 8Day 0
WTIl33-/-
0
2
4
6
8
%G
ATA-
3+ (IL
C)
Day 8Day 0
0
2
4
6
8
%G
ATA-
3+ (T
cel
ls)
Day 8Day 0
WTIl33-/-
*
0
2500
5000
7500
10000
T H2
per c
olon
Day 8Day 0
WTIl33-/-
*
A
B
D
CD
45
FSC-A
CD
90
CD19 C
D12
7 Lin-2
T cells
ILC
ILC WT Il33-/-
CD
90
2.3 3.8
GATA3
T cells WT Il33-/-
CD
90
GATA3
2.3 7.0
C
! 8
114!Supplementary figure 6. There is no defect in induction of anti-inflammatory and 115!epithelium healing genes or T regs in the colons of Il33-/- mice. (A-C) Quantification of 116!expression of indicated genes in the colons of WT and Il33-/- deficient mice at indicated days 117!post DSS administration by qRT-PCR. (D) Quantification of T regulatory cells gated as CD19-118!CD3+CD4+Foxp3+ cells in the lamina propria of the colon by flow cytometry. Data represent two 119!independent experiments and analyzed by Kruskal-Wallis test followed by Dunn’s post test. 120!Error bars represent mean±S.E.M with 5 mice per group per time point. 121!
122!
123!
124!
125!
126!
127!
128!
129!
130!
131!
132!
133!
134!
135!
048
300400500
Lipo
calin
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 4
0
2
4
6Il2
2 lo
g 10
Rel
ativ
e ex
pres
sion ****NS NS
WTIl33-/-
Day 0 Day 8Day 40
5
10
15
Reg
3βR
elat
ive
expr
essi
on NS NS NS
Day 0 Day 8Day 4
0.00.51.01.52.02.5
Occ
ludi
nR
elat
ive
expr
essi
on NS NSNS
Day 0 Day 8Day 40.0
0.5
1.0
1.5
2.0
Zo1
Rel
ativ
e ex
pres
sion **NS NS
Day 0 Day 8Day 4
0
5
10
15
Reg
3γR
elat
ive
expr
essi
on **NS NS
Day 0 Day 8Day 4
012345
Mpt
xR
elat
ive
expr
essi
on NS NSNS
Day 0 Day 8Day 40
1
2
3
4
S100
A9R
elat
ive
expr
essi
on NS NS ***
Day 0 Day 8Day 4
10
20
30
40
50
CD
4+ %
Fox
p3+ **
Day 0 Day 8Day 4
NS **
A
B
C D
! 9
136!Supplementary figure 7. Increasing Akkermansia level in WT mice increases colitis 137!susceptibility. (A) Q-PCR analysis of indicated bacteria from stool samples of WT and Il33-/- 138!mice 1-week administration of water or broad-spectrum antibiotic cocktail (125 mg/l 139!ciprofloxacin, 1 g/l bacitracin, 2 g/l streptomycin, 1.5 g/l metronidazole and 172 mg/l gentamycin) 140!in their drinking water. (B) IL-1α measurement in colon explants at day 4 post DSS 141!administration (C) Body weight loss and (D) disease activity index of mice during DSS 142!administration. (E) Colon length measurements and (F) representative colon images at day 8 143!post DSS administration. Data represent two independent experiments and analyzed by 144!Kruskal-Wallis test (A) (B) and (D) or two-way ANOVA (C) followed by Dunn’s post test. Error 145!bars represent mean±S.E.M. and each dot represents an individual mouse. N=10 mice for WT 146!and water, Il33-/- and water group, 5 for WT and antibiotics group. 147!
148! 149!
150!
151!
152!
153!
154!
155!
156!
157!
158!
159!
160!
161!
4
6
8
10
Col
on le
ngth
(cm
) ********
Water Abx
0.0
0.5
1.0
1.5
Bac
tero
ides
Rel
ativ
e to
WT
NS NS
Water Abx0.0
0.5
1.0
1.5
2.0
Pre
vote
lla R
elat
ive
to W
T
*NS
Water Abx
0 3 6 7 970
80
90
100
110
Days post DSS
Initi
al b
ody
wei
ght (
%)
Il33-/-, Water WT, Water ****
WT, Abx
0
2
4
6A
kker
man
sia
Log 10
Rel
ativ
e to
WT
********
Water Abx
WT Il33-/-
0
100
200
300
400
IL-1α
(pg/
ml)
******
Water Abx
Water WT Il33-/- WT
Abx
A B
C D E
! 10
162!Supplementary figure 8. Il33-/- littermates have decreased intestinal IgA, increased level of 163!Akkermansia and susceptibility to colitis. (A) IgA measurement by ELISA and (B) qRT-PCR 164!analysis of indicated bacteria from stool samples of WT, Il33+/- and Il33-/- mice 4 weeks after 165!separation. (C) Body weight loss of mice during DSS administration. (D) Colon length 166!measurements and (E) representative colon images at day 8 post DSS administration. Data is 167!analyzed by Kruskal-Wallis test (A) (B) and (D) or two-way ANOVA (C) followed by Dunn’s post 168!test. Error bars represent mean±S.E.M. and each dot represents an individual mouse. N=11 169!mice for WT, 14 for Il33+/- and 6 for Il33-/- group. 170!
171!
172!
173!
174!
175!
176!
177!
178!
179!
180!
181!
WT Il33+/- Il33-/-0.0
0.3
0.6
0.9
1.2In
test
inal
IgA
(uni
ts/m
g)**
*
WT Il33+/- Il33-/-0
1
2
3
4
Akkermansia
Rel
ativ
e to
WT ****NS
0 3 5 6 7 9
90
100
110
Days post DSS
Initi
al b
ody
wei
ght (
%)
WTIl33+/-
Il33-/-
** ****
WT Il33+/- Il33-/-0.0
0.5
1.0
1.5
2.0
Bacteriodes
Rel
ativ
e to
WT
NS NS
WT Il33+/- Il33-/-6
7
8
9
10
11
Col
on le
ngth
(cm
) *****NS
WT Il33+/- Il33-/-
A B
C D E
! 11
Supplementary Methods 182!
Preparation of single cell suspension from colon. 183!
Single cell suspension was prepared from the colon as described previously (1). Briefly, 184!
for removal of epithelial cells, the colon was washed, cut into small pieces, and then the 185!
pieces were incubated with calcium- and magnesium-free HBSS supplemented with 5% 186!
FBS and 5 mM EDTA (Sigma-Aldrich) at 140 rpm at 25°C for 30 min. The tissues were 187!
then incubated with RPMI 1640 containing 10% FBS and 0.5 mg/ml collagenase type IV 188!
for 1 hour at 37°C with shaking at 150 rpm. The liberated cells were collected by 189!
passage through a 70 µm nylon mesh. The isolated cells from the EDTA (epithelial) and 190!
collagenase (lamina propria) treated fractions were separated on a 40/80% 191!
discontinuous Percoll gradient (GE Bioscience). The following monoclonal antibodies 192!
were used in appropriate combinations: anti-CD3 (clone 145 – 2C11), anti-CD4 (clone 193!